Cytokinins are plant hormones that mediate cell division and development. This group of hormones was discovered by Miller et al. (Miller et al., Journal of American Chemical Society 77:1392, 1955; Miller et al., Journal of American Chemical Society 78:1375–1380, 1956) with the identification of the first synthetic cytokinin, kinetin. The first naturally occurring cytokinin, zeatin (trans-zeatin), was discovered by Letham (Letham et al., Ann. Botany 41:261–263, 1976) in corn, and the structure of zeatin was determined by Shaw and Wilson (Shaw et al., Proceedings of Chemical Society 231, 1964). Zeatin is the most active and ubiquitous cytokinin in all plant species examined to date. Other naturally occurring cytokinins are structurally related to zeatin (Shaw, Cytokinins, Chemistry, Activity and Function, Mok and Mok, CRC Press, 15–34, 1994).
The critical importance of cytokinins in plant development was illustrated by the classic tissue culture experiments of Skoog et al. (Skoog et al., Science 148:532, 1965). These experiments established that plant cell division requires cytokinin. Furthermore, the ratio of cytokinins to auxins (another group of plant hormones) was shown to indicate whether undifferentiated plant cells would develop into shoots (high cytokinin to auxin) or roots (low cytokinin to auxin), or continue to proliferate as callus tissues (intermediate cytokinin to auxin ratio). Thereafter, cytokinin was found to be involved in every phase of plant growth (Mok, Cytokinins, Chemistry, Activity and Function, Mok and Mok, CRC Press, 155–166, 1994). In general, cytokinins have growth promoting effects, from seed germination and shoot development to retarding senescence and increasing fruit and seed set
The effects of cytokinins in controlling plant growth have been extensively utilized in plant tissue culture to micropropagate and clone plants and to regenerate whole plants from cells of many species (Krikorian, Plant Hormones-Physiology, Biochemistry and Molecular Biology, 2nd Edition, Davies, Kluwer Academic Publishers, 774–796, 1995). In fact, the application of cytokinins in vitro contributes significantly to advances in plant biotechnology. In agricultural applications, external applications of cytokinins on whole plants are used to obtain enhanced fruit set and gram yield of food crops and longer shelf life of ornamentals (Hradecka et al., Physiology and Biochemistry of Cytokinins in Plants, Kaminek et al., SPB Academic Publishing, 245–247, 1992; Karanov et al., Progress in Plant Growth Regulation, Karssen et al., 842–851, 1992; Lewis et al., Physiol. Plant. 98:187–195, 1996; Minana et al., J. Exp. Bot. 219:1127–1134, 1989).
In whole plants, cytokinins are synthesized in the roots and transported to above ground parts (Letham, Cytokinins: Chemistry, Activity and Function, Mok, and Mok, CRC Press, Boca Raton, 57–80, 1994), although other actively growing tissues also have biosynthetic capacity. Two biosynthetic pathways have been proposed for cytokinin biosynthesis. The first is the direct pathway, involving formation of N6-isopentenyladenosine phosphate from AMP and dimethylallyl pyrophosphate, followed by hydroxylation of the side chain to form zeatin-type compounds. The second pathway is the indirect pathway, in which cytokinins are released by turnover of tRNA containing cis-zeatin (Prinsen et al., Plant Growth Regul. 23:3–15, 1997). Plant AMP isopentenyltransferases have not been found in spite of the identification of such genes from bacteria such as Agrobacterium tumefaciens (Akiyoshi et al., Proc. Natl. Acad. Sci. USA 81:5994–5998, 1984; Barry et al., Proc. Natl. Acad. Sci. 81:4776–4780, 1984; Beaty et al., Mol. Gen. Genet. 203:274–280, 1986). In fact, plant DNA homologous to these bacterial genes has not been reported. Therefore, the intermediates, the enzymes, and the genes involved in direct pathway(s) of cytokinin biosynthesis in plants remain unproven or unknown. Cytokinins occur adjacent to the anticodon in tRNAs recognizing codons beginning with U (Skoog et al., Ann. Rev. Plant Physiol. 21:359–384, 1970; Taller, Cytokinins, Chemistiy, Activity and Function, Mok and Mok, CRC Press, 101–112, 1994). The indirect pathway involves release of cytokinins from breakdown of such tRNA. Although the weakly active cis-zeatin is the major cytokinin in plant tRNA, cis-zeatin can be converted to trans-zeatin by cis-trans isomerization (Bassil et al., Plant Physiol. 102:867–872, 1993).
Although trans-zeatin and its derivatives are prevalent in most plants, cis-zeatin has been found in potato (Mauk et al., Plant Physiol. 62:438–442, 1978), tobacco (Tay et al., Plant Sciences 43:131–134, 1986), rice (Izumi et al., Plant Cell Physiol. 29:97–104, 1988), and as the predominant cytokinin in chickpeas (Emery et al., Plant Physiol. 117:1515–1523, 1998). Relatively high levels of cis-zeatin occurs in below-ground parts of the plants such as roots and tubers. Cones of hops (Watanabe et al., Plant and Cell Physiol. 22:489–500, 1981) and unisex flowers of Mercurialis (Durand et al., Cytokinins, Chemistry, Activity and Function, Mok and Mok, CRC Press, 295–304, 1994) also contain much cis-zeatin. Therefore, cis-zeatin may play a unique role in biosynthesis as well as in mediating specific developmental steps not yet discovered.
Cytokinins are converted to various metabolites in plant tissues (Jameson, Cytokinins, Chemistry, Activity and Function, Mok and Mok, CRC Press, 113–128, 1994). For example, the metabolites of zeatin include O-glycosylzeatin, N-glucosylzeatin, zeatin riboside, and zeatin nucleotides. The precise functions of these metabolites are still uncertain. However, some may be the stored or the transported form of the active compound, zeatin. O-Glucoside of zeatin (FIG. 1) may be such a metabolite (Badenoch-Jones et al., Plant Cell and Environment 19:504–516, 1996). Trans-Zeatin O-glucoside was first discovered by Letham et al. (Letham et al., Ann. Botany 41:261–263, 1976) and has been found in all crops examined including corn, beans, poplar, soybean, etc. As O-glucosylzeatin can be readily converted back to its active form, zeatin, by the removal of the glucose moiety (via the action: of wide-spread enzymes, β-glucosidases), O-glucosylzeatin is considered a reversible reserve of active cytoknin (Brzobohaty et al., Science 262:1051–1054, 1993). Also, O-glucosylzeatin is resistant to attack by cytokinin oxidases (McGaw et al., Planta 159:30–37, 1983) that degrade the parent compound, zeatin. Therefore, O-glucosylzeatin may be important in cytokinin action by serving as an interchangeable reserve and as an oxidase resistant form of zeatin. Another metabolite, zeatin O-xyloside was fist discovered in beans (Phaseolus) by Lee et al., (Lee et al., Plant Physiol. 77:635–641, 1985). Zeatin O-xyloside is also resistant to degradation and can be reconverted to zeatin. Two enzymes, zeatin O-glucosyltransferase (ZOG) and zeatin O-xylosyltransferase (ZOX), catalyzing the formation of zeatin to O-glucosylzeatin and O-xylosylzeatin, respectively, were first purified and characterized in the Mok laboratory (Turner et al., Proc. Natl. Acad. Sci. 84:3714–3717, 1987; Dixon et al., Plant Physiology 90:1316–1321, 1989). The occurrence of the enzymes is species specific. The former was isolated from lima beans (Phaseolus lunatus) and the latter from common beans (P. vulgaris). The isolation of the enzymes was followed by the generation of specific antibodies recognizing the enzymes (Martin et al., Plant Physiology 94:1290–1294, 1990). Subsequently, two genes encoding the respective enzymes were cloned (Martin et al., Proc. Natl. Acad Sci. USA 96:284–289, 1999; Martin et al., Plant Physiol. 120:553–557, 1999). The genes were designated as ZOG1 (for zeatin O-glucosyltransferase) and ZOX1 (for zeatin O-xylosyltransferase).
Plants having modified endogenous zeatin activity would be of significant agricultural importance. Such plants could be created through genetic engineering if the genes regulating zeatin were available. It is to such genes, and polypeptides encoded thereby, that the present disclosure is directed.